The present invention relates to skin protection agents, in particular to topical compositions which can prevent the passage of toxic chemicals through the skin, such as barrier creams.
There are many occasions where people are exposed to chemicals or agents which have some degree of toxicity and which may penetrate the skin. It is often desirable or necessary to prevent contact of these chemicals to the skin as far as possible.
For instance, many volatile pesticides can provide a potential hazard to operators applying them. Examples of such pesticides include volatile insecticides, particularly organophorphorus insecticides such as isofenphos, or diethyl toluimide. In other industrial applications, workers exposed to industrial solvents such as acetone, chloroform, methanol, hexane, benzene and toluene, may require protection.
In chemical warfare situations, highly toxic volatile chemicals, which may penetrate the skin can be used. These include Sarin, Soman and Tabun as well as sulphur mustard and Lewisite.
Sulphur mustard (SM) is a vesicating chemical used as a war gas. It is a potent alkylating agent which is thought to be toxic to living tissue by virtue of its ability to alkylate vital cellular constituents (Fox M. and Scott M., Mut. Res (1980) 75, 131-168). SM has been shown to alkylate DNA. RNA and proteins (Paprimeister B. et al., (1991) Medical Defence against Mustard Gas (CRC Press) 91-122), though a causal link between alkylation of either of these cellular constituents and vesication has yet to be established.
Although considerable protection from toxic chemical vapours can be afforded by protective clothing in the form of respirators, charcoal cloth garments and gloves these measures may not be completely effective in all circumstances. The use of a barrier cream might augment the charcoal cloths used in protective clothing and permit tasks requiring manual dexterity to be carried out in an atmosphere containing chemical agent vapour, without gloves for short periods.
Traditional barrier creams have placed a passive barrier between the skin and the environment which prevent access of chemicals generally to the surface of the skin. Cream bases consist of an emulsion, either oil in water or water in oil (Barry B. W (1983) Drugs Pharmaceutical Sci 18 296-350). Water in oil emulsions are unsuitable as barrier creams because the oily deposit they leave on the surface of the skin results in loss of tactility. On their own oil in water type creams have required layers of greater than 0.56 mm to be established and maintained on the skin in order to be effective. More recently creams based on silicone derivatives have allowed effective barriers against general chemical penetration of the skin to be produced using very thin layers of cream (EP-A-0401840, Japanese Patent Appln no. 57-26610).
Formulation of a reactive chemical which reacts with specific target chemical groups in the cream converts a passive barrier cream into an active cream which sequesters and inactivates a specific group of toxic chemicals before they can reach the living layers of the skin.
The use of reactive molecules which would inactivate SM before it could react with cell constituents has been investigated. A series of sulphydryl compounds designed to enter the living cell and fortify it against reactive compounds such as SM has already been disclosed in PCT GB91/01462 and GB90148994.5.
It is known that increased levels of GSH and GSH synthetic enzymes (Evans et al., Cancer Res. 47 2525-2530, Ahmed S. et al., J. Biol. Chem. 262 15048-15053, and Ahmed S. et al., J. Cellular Physiol. 131, 240-246) confer resistance to nitrogen mustard on cell cultures. In previous studies attempting to protect cell cultures against SM, Gross C. L. and Smith N. J. (Proc. 1993 Med. Def. Bioscience Rev. (US Army Medical Research and Development Command) 1 141-147) found that human lymphocytes pretreated with 10 mM L-oxothiazolidine-4-carboxylate for 48 hours and subsequently exposed to 10 xcexcM SM were 20% more viable than SM only treated cells 48 hours after exposure. Other attempts to protect human lymphocyte cultures (Gross et al. Cell Biology and Toxicology (1993) 9 259-268) by raising intracellular levels of GSH using N-acetyl cysteine (10 mM) and subsequently challenging with 10 xcexcM SM increased viability by only 15 to 19% of control cultures at 48 hours.
The stratum corneum is the dead layer of skin which acts as a barrier to chemical penetration through the skin (Tregear R. T. (1966) Theoretical Exp. Biol. 5, 21-22). The stratum corneum is composed of the dead xe2x80x9cprotein ghostsxe2x80x9d of the living cells of the epidermal layer of the skin, surrounded by a unique mixture of lipids which the cells of the epidermis produce (Wertz P. W. et al., (1989) xe2x80x9cStratum corneum: Biological and Biochemical considerationsxe2x80x9d in Transdermal Drug Delivery, Ed Hadgraft J and Guy R A. 1-22).
HMT has been used prophylactically to protect rabbits and man against exposure to lethal phosgene doses (Diller W. F. J. Occ. Med (1978) 20 189-193).
The present application relates to a topical composition for use particularly against SM. In particular the invention relates to an active barrier cream for SM.
The present invention provides a composition which comprises a composition for topical application which comprises hexamethylene triamine or analogues or derivatives thereof.
HMT has the following structure (I) 
It is believed that because the structure of HMT contains four nucleophilic nitrogen groups, with a similar electronic structure to N-7 in guanine, it neutralises the bifunctional SM more effectively than conventional monofunctional thiol ligands.
Suitable analogues of HMT are compounds which have organic moieties conjugated to the HMT cage structure for example of formula (II) 
where R1 is an organic group and X is a anion.
The HMT moiety will hereinafter be represented as #.
Examples of suitable groups for R1 include optionally substititued straight or branched hydrocarbon chains such as alkyl chains, having from 1 to 50 carbon atoms, for example from 6 to 32 carbon atoms. In particular, the hydrocarbon chains are optionally substituted by one or more substituents selected from aryl, such as phenyl, optionally substituted by halogen, and/or halogen atoms, such as fluorine, chlorine or bromine and particularly fluorine. A particular group R1 is a fluorobenzyl or a group (CH2)nCH3 where n is from 8 to 20, suitably about 17.
Further examples of such compounds include compounds of the following formulae:
(#)xe2x80x94CH2(CH2)22CH3 
(#)xe2x80x94CH2(CH2)14CH3 
where # represents a hexamethyltetraminyl group.
Suitable anions X are chosen such that the compound of formula (II) are pharmaceutically acceptable salts, in particular those which are suitable for topical application, for example halide ions such as iodides or bromides.
Suitable derivatives of HMT are made by the conjugation of HMT with either the normal constituents of barrier creams (such as palmitic or stearic acids), the normal constituents of the stratum corneum (cholesterols or ceramides) or large long chain aliphatic molecules. Suitably the long chain aliphatic molecules have from 16 to 32 carbon atoms. Particular aliphatic molecules which may be employed are fatty acids having from 16 to 32 carbon atoms.
Analogues of formula (II) are suitably prepared by reacting HMT with a compound of formula (III)
Yxe2x80x94R1 xe2x80x83xe2x80x83(III) 
or a salt thereof; where R1 is as defined in relation to formula (II) and Y is a leaving group, and thereafter if necessary converting a salt produced to a different salt.
Suitable leaving groups for Y include halogen such as fluorine, chlorine or bromine, particularly bromine, as well as mesylate and tosylate. The reaction is suitably effected in an organic solvent, for example chloroform, alcohols such as methanol or ethanol, acetonitrile at elevated temperatures conveniently at the reflux temperature of the solvent.
Salts of formula (III) may be any salt as convenient depending upon the particular nature of the reagents involved. They may subsequently be changed to a different pharmaceutically acceptable salt using ion exchange procedures, for example by passing the compound down an ion exchange column as is conventional in the art.
Suitably the composition further comprises a cream base.
These compositions place an active barrier between the cellular constituents of the skin and the outside world, at the level of the dead layer of the skin, the stratum corneum. This is achieved by a molecule which reacts with and sequesters SM which is suitably fixed on the surface of the strateum corneum in a very thin layer using a barrier cream base.
Suitably the composition contains further active reagents, in particular other nucleophilic scavengers. Such groups are useful because SM reacts readily with a wide range of nucleophilic groups found in biological systems. The most important of these are thought to be the N-7 groups of guanine residues in nucleic acids and the sulphydryl groups of cysteine containing peptides and proteins, the affinity of SM for the former biological ligand in particular, dictates the necessity of having protective measures in place prior to SM exposure. Since SM is bifunctional it can crosslink biomolecules, forming DNA crosslinks between guanine residues (Papirmeister B et al., Medical Defence against Mustard Gas (CRC Press Inc. Bocca) (1991) pp106).
A preferred group of further reagents for use in the composition of the invention include compounds which have nucleophilic nitrogen and/or sulphur atoms, preferably nitrogen.
Examples of sulphur containing nucleophilic compounds include glutathione (GSH) and esters of cysteine, for example as shown in WO 92/04024.
When two different species of nucleophile (ie N and S) are used as extracellular scavengers of SM, they unexpectedly have had an increased protective effect in this study, compared to using the compounds singly. Thus a composition which comprises both nucleophilic nitrogen and sulphur are preferred. Such compositions include combinations of HMT or derivatives or anlogues and GSH. Alternatively, thiolated forms of the conjugating agents mentioned above, such as thiol derivatives of cholesterol or a ceramide may be used.
In addition, it has been shown that the use of ligand mixtures increases the capacity of protection media to reduce SM-induced cytotoxicity without synergistic cytotoxic effects.
Small molecules in a barrier cream can diffuse through the skin and be absorbed into the blood which may result in local irritation or systemic toxicity. Therefore, in a preferred embodiment, the cream includes large lipophilic molecules which do not penetrate the skin leaving the reactive molecule either on the surface of the skin or retained within the superficial layers until they slough off in the normal cycle of the epidermal tissue.
Consequently in a preferred embodiment, the active barrier cream of the invention is prepared by forming a complex comprising a suitable reactive molecule and a large lipophilic molecule and the resultant complex is formulated into a barrier cream base.
A specific embodiment of the invention is a reactive barrier cream which comprises HMT in an oil in water emulsion cream base.
However, it has been found that a particularly advantageous carrier for the compositions comprises a perfluorinated polymeric compound.
Such compounds have a low surface energy and thereby prevent partitioning of the volatile chemical into the topical composition and skin. Indeed it has been found that such compounds can act as effective barrier creams when used alone and this forms a further aspect of the invention.
Suitably the perfluorinated polymeric compound is of formula (IV):
CF3Oxe2x80x94[CF(CF3)CF2O]nxe2x80x94[CF2O]mxe2x80x94CF3 xe2x80x83xe2x80x83(IV) 
where n and m are independently selected from 4 to 150, suitably from 6 to 140.
Suitably the compound of formula (IV) is in the form of an oil. Examples of such compounds are set out in the following Table:
These compounds are available commercially from Aldrich Chemical Company, Gillingham, Dorset and are sold under the trade name xe2x80x9cFomblinxe2x80x9d(trademark). Other compounds of formula (IV) may be prepared using conventional methods.
The perfluorinated compounds produce a significantly improved barrier to SM than conventional oil-in-water emulsions, which under some conditions, may actually trap the SM and increase the penetration rate.
The compositions of the invention comprise a substantial portion of perfluorinated polymeric compound for instance up to 100% w/w perfluorinated compound. Suitably, the compositions comprise from 30-100% w/w perfluorinated compound and preferably in excess of 50% w/w. Where 100% prefluorinated compound is used, this must comprise an oil.
These compositions may further comprise active agents other than or in addition to HMT or analogues or derivatives thereof as described above, which react with or sequester the chemical which it is intended should be prevented from reaching the skin.
For instance, reactive molecules which would inactivate for example, SM before it could react with cell constituents may be included. Such compounds include compounds disclosed in PCT GB91/01462 and GB90148994.5 or WO 92/04024 as mentioned above. A particular reactive agent which may be used in the formulations of the invention is potassium butadione monoximate.
Where present, preferred ratios of the active compound: perfluorinated compound in a barrier cream formulation are, as before, from 5:95 to 60:40 w/w for instance from 10:90 to 60:40 w/w, most preferably 25:75 w/w.
The compositions may be in the form of oil in water or water in oil emulsions. Other components of such emulsions include emulsifying agents and oils conventionally used in barrier cream type formulations such as mineral or silicone oils. However, the compositions are preferably oils rather than emulsions.
Preferably, the compositions of the invention further contain lower molecular weight perfluorinated compounds such as polytetrafluoroethylene. These compounds can affect the viscosity of the formulation, making them more effective barriers. Suitable compounds of this type are available from ICI plc. They have a molecular weight of the order of 106, and a particle size of approximately 6microns.
The present inventors have studied the ability of HMT to protect cells against the toxic effects of SM in culture and to prevent the penetration of SM through isolated skin, to assess the feasibility of its prophylactic use to prevent SM induced skin injury. Penetration of radiolabelled SM across human stratum corneum has been measured in vitro using glass diffusion cells.
In the cell culture study, Simian virus keratinocyte-14 (SVK-14xe2x80x94a human keratinocyte line transformed with the SV40 virus) cell cultures were used to assess the protection afforded by a prophylactic mixture of sulphur and nitrogen containing compounds against SM, and the effect of using the compounds singly. Mitotically active cells are known to be more sensitive to the effects of DNA alkylating agents such as SM (Fox M. and Scott M. et al., Mut. Res. 75 131-168), since the formation of cross-linked DNA lesions prevents mitosis, leading to unbalanced metabolism and cell death (Cohen et al., Proc. NY Nat. Acad. Sci. 40 885-893).
The use of an actively mitosing, keratinocyte-derived cell line as used in the present study has several important advantages over resting peripheral blood lymphocytes used in the studies of Gross et al. (supra): firstly, the epithelial morphology of the SVK culture means that it is a better model of the main targets of SM intoxication, namely the basal epidermis, the broncho-epithelium and the corneal epithelium; secondly, SVKs are an attached cell-line, enabling the study of specific intercellular interactions following SM exposure; finally, because the SVK cultures are mitotically active they will be more susceptible to SM for the reasons outlined above.
Altogether, the use of SVK cultures represents a more severe test of the efficacy of putative prophylactic regimes than does the use of peripheral blood lymphocytes. Consequently, the level of protection of the SVK cultures observed in this study is far greater than that achieved in previous studies aimed at increasing intracellular GSH levels.
In the present tests, assessment of the pretreated cultures 96 hours after exposure to SM demonstrated a significant increase in the viability of the SVK cells compared to those exposed to SM. Culture viability was measured using the NR assay. This involves the uptake of NR dye into intracellular vesicles such as lysosomes and gives a measure of cellular activity as well as membrane integrity.
63.3% of cells in cultures pretreated with 4 mM GSH/10 mM HMT remained viable compared to 33.5% of SM exposed cells, this was mirrored in the photomicrographs (not included) showing SM exposed and pretreated cultures respectively. SM exposed cells have taken up the dye but the drastically altered cellular morphology (extended cytoplasm and congregation of lysosomes around the swollen nucleus) indicates that these cells are not in a truly viable and proliferative state. In comparison, the pretreated cultures show protection of the cells with a preserved monolayer, a great reduction in the number of hyperplasmic cells and a more diffuse distribution of the lysosomes in the cytoplasm.
Cell numbers were measured using crystal violet staining of DNA. Pretreatment of the SVK cultures with 4 mM GSH/10 mM HMT did not significantly alter the growth rate compared to the control, however the difference between pretreated cultures compared to those exposed to SM is marked. Not only is there a preservation of cell numbers at every time-point but also the rate of culture depopulation is less and there is an eventual recovery of the population by 96 hours at a comparable growth rate to the control. The use of either 8 mM GSH or 10 mM HMT singly was not as effective as using the compounds in combination at the 72 hour time-point, with increases in viability of 19.8% or 22.6% compared to 29.8%. The use of 16 mM GSH gave an increase in viability of 20.6% at 72 hours compared to the SM control (p less than 0.001), whilst the use of 20 mM HMT did not alter the toxicity of SM (p greater than 0.05) at 72 hours. The use of GSH and HMT in combination confered greater protection on the cultures than higher concentrations of the single prophylactics.
Penetration of radiolabelled SM across rubber latex membranes and human epidermal skin membranes has been measured in vitro using glass diffusion cells is also reported hereinafter.
In a diffusion cell system the chemical diffuses through the membrane (the penetrant) by partitioning between its vehicle and the membrane, then between the membrane and the receptor fluid. When the thermodynamic activity, of the penetrant (usually synonymous with concentration) on both sides of the membrane is equal the system is in equilibrium and there will be no further change in the amount of penetrant in the receptor fluid.
In certain diffusion studies reported hereinafter, the ability of each composition to slow the rate of penetration through the membrane was shown by a calculation of the retardant index (RI) using the equation
RI=Jmax(control)/Jmax(treated) 
where Jmax is the maximum penetration rate of sulphur mustard through control (untreated) and treated membranes.
When a compound reacts with the penetrant on the surface of the membrane and forms a product which does not penetrate the membrane, the total amount of penetrant in the receptor fluid at equilibrium will be reduced. In such a case, and if the penetrant is applied as an undiluted liquid the measured maximum rate of penetration would only be reduced if the presence of the product reduces the thermodynamic activity of the penetrant.
When using radiolabel to measure the amount of penetrant in the receptor chamber no distinction can be made between unreacted penetrant and product. Therefore if the product also penetrates the membrane the measured maximum diffusion rate and total amount penetrating will be increased.
When the reactive protectant is applied to the membrane in a cream base the penetrant must partition into the cream and then into the membrane in order to enter the receptor chamber. The partitioning of the penetrant into the cream can thus modify the rate of penetration by altering the partitioning of the penetrant into the membrane. The reactive protectant must also retain its reactivity with the penetrant within the chemical environment of the cream base.
In diffusion studies reported hereinafter, the reduction in the total amount of SM penetrating the epidermal membranes pretreated with HMT alone, without effect on the maximum flux rate, supports the hypothesis that HMT is reacting with SM to form a product which does not cross the membrane.
Mixing HMT with oil in water cream bases retains the reduction in total amount penetrated indicating that any reaction also occurs in this medium. The reduction in maximum flux rate observed in a cream base, such as Stokoderm(copyright) obtainable from Arco Skin Care Products, Hull, may result from a reduction in partitioning of SM into the epidermal membranes from the cream. The possibility that the product of any reaction may partition into the membrane from the cream base cannot be discounted and may explain the slight increase in maximum penetration rate measured through the oil in water emulsions containing beeswax and brij 52.
In summary, the reduction in the cytotoxicity of SM and its penetration of epidermal membranes in the presence of HMT support the use of HMT or one of its analogues as a reactive constituent of barrier creams.
As illustrated hereinafter, formulations referred to in this application can reduce the rate of penetration of SM through isolated stratum corneum by five fold, and sometimes up to 45 fold, and reduce the total amount penetrated by up to 90%.